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| Status |
Public on Feb 19, 2019 |
| Title |
AB2729 (scRNA-Seq) |
| Sample type |
SRA |
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| Source name |
Auricular lymph node
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: Lymph node
cell type: innate immune cells
selection marker: CD45+, CD19-, CD3-, TCRb-
infection: Nippostrongylus brasiliensis
time point: day1
treatment(1): Intradermal injection of inactivated pathogen
treatment(2): N/A
replicate id: Nb day1 rep.3
mouse age: 8 weeks
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| Treatment protocol |
[treatment protocol (1)] Mice were anesthetized and the indicated inactivated pathogens were administered by intradermal injection into the ear pinna. PBS was injected into the ear pinna of control animals.
[treatment protocol (2)] mice were treated with depleting or neutralizing antibodies or their isotype controls: Anti-NK1.1 (200 µg/dose), anti-IFNγ (500 µg/dose), Mouse IgG2a Isotype control and Rat IgG1 Isotype control were injected 24 hours prior to immunization and on the day of immunization
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| Growth protocol |
6 to 8 weeks-old mice housed at the Weizmann Institute animal facility.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mice were euthanized and auricular LNs were disected. Lymph nodes were digested in IMDM containing 100µg/mL Liberase TL and 100µg/mL DNase I for 20 minutes at 37°C. In the last 5 minutes of incubation, EDTA was added at a final concentration of 10mM. Cells were collected, filtered through a 70µm cell strainer, washed with IMDM and maintained strictly at 4°C. Cells were stained with FACS antibodies, then sorted into 384-well plates
Single-cell RNA-seq libraries were prepared as previously described {Jaitin, 2014}. In brief, mRNA from single cells sorted into capture plates were barcoded and converted into cDNA and then pooled using an automated pipeline. The pooled sample was linearly amplified by T7 in vitro transcription, and the resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with pool barcodes and Illumina sequences during ligation, RT, and PCR. Each pool of cells was tested for library quality and concentration as described previously {Jaitin, 2014}
single cell RNA-seq for gene expression quantitation
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Note: Second read contained only cell and molecule barcodes. This information was appended to the fastq entry header
bcl2fastq/2.15.0.4
Sequences with RMT of low quality (defined as RMT with minimum Phred score of less than 27) were filtered out.
Pool-barcode and well-barcode-RMT were extracted from the first and second end of the read (respectively) and concatenated to the fastq header, delimited by a underscore i.e. POOL_BARCODE_WELL_BARCODE_RMT while "NNNNNN" was used as a place holders if plate barcode was not used.
Reads were separated by POOL_BARCODE_WELL_BARCODE header data, allowing 1 sequencing error. This process created a single fastq file for each source well.
Genome_build: mm9
Supplementary_files_format_and_content: [AB*.txt] tab-delimited text files include mRNA molecule count values for each Sample
[metadata.txt] file associating each single cell with its amplification batch and index sorting readouts
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| Submission date |
Jan 14, 2019 |
| Last update date |
Feb 19, 2019 |
| Contact name |
Ido Amit |
| E-mail(s) |
ido.amit@weizmann.ac.il
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| Phone |
972-8-9343338
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| Organization name |
Weizmann Institute of Science
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| Department |
Immunology
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| Street address |
234 Herzl st.
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| City |
Rehovot |
| ZIP/Postal code |
760001 |
| Country |
Israel |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE125044 |
Single cell analysis of diverse pathogen responses defines a molecular roadmap for generating antigen-specific immunity |
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| Relations |
| BioSample |
SAMN10741651 |
| SRA |
SRX5248384 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3561693_AB2729.txt.gz |
615.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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